α-Sialyl-oligosaccharides of formula (I)
wherein R is a mono- di- or oligosaccharide residue with free hydroxyl groups are present in mammals and birds tissues and in predominant form of lipooligosaccharides, lipopolysaccharides or glycans of glycoproteins. They exist in a variety of glycosidic bonds, more typically α(2-3) and α(2-6) galactose (or lactose). The function of these sialosides varies greatly in animals according to the structural heterogeneity of the oligosaccharide portion. They are mediators of inter and intra-cells events in particular play an important role in the physiology and growth of many pathogen agents (D K Ress, et al., Current Organic Synthesis, 2004, 1, 31-46).

One liter of human milk contains about 5-10 g of free oligosaccharides, this content is similar to the content of proteins and exceeds the lipid content. More than 130 different oligosaccharides were identified in human milk (Human Milk Oligosaccharides—HMO), formulations of artificial milk for babies derive from bovine milk and contain only trace amounts of these oligosaccharides that are specific of the human species. The fundamental building blocks of oligosaccharides of human milk are the 5 monosaccharides D-glucose (Glc), D-galactose (Gal), N-acetylglucosamine (GlcNAc), L-fucose (Fuc) and sialic acid (N-acetyl-neuraminic acid, Neu5Ac). The terminal reducing end can be formed by lactose (Galβ1-4Glc) or more repetitive units (up to 15 unit) of N-acetyllactosamine (Galβ1-3/4GlcNAc). Lactose or polylactosamine may be sialylated with α2-3 and/or α2-6 bonds. Examples of sialosides of human milk are: 3′-sialyl-3-fucosylactose (3′S3FL), 6′-sialyllactose (6′SL), 3′-sialyllactose (3′SL), 3′-sialyllattosamine (3′SLN), 6′-sialyllactosamine (6′SLN).
Among sialosides mainly present in mammalian tissues and in human milk the compound of formula (Ia) 6′-sialyllactose (N-acetylneuraminyl-lactose, α-NeuNAc-(2→6)-β-D-Gal-(1→4)-D-Glc or 6′-SL) is of particular importance because it is an important constituent of glycoproteins and glycolipids involved in various cell pathway events including cell recognition and immune response. The 6′-SL and their salts are interesting as supplements in food formulations for infants. As for the salts of the 6′-sialyllactose in literature only the sodium salt (CAS Number: 157574-76-0; FW: C23H38NO19Na, 6′-sialyllactose sodium salt, 6′-N-Acetylneuraminyl-lactose sodium salt) and the ammonium salt are known. While the sodium salt is acceptable for food and pharmaceuticals, the ammonium salt is potentially toxic because of the ammonium ion. For this reason it is necessary to get 6′-SL in alternative salt forms to the known ones that may be acceptable for food and pharmaceuticals.
At the state of the art various strategies are known for synthesis of sialyl-oligosaccharides (including 6′SL) and all foresee a convergent approach in which the sialic activated fragment (donor) is regio- and stereo-selectively bound to the oligosaccharide portion (acceptor). For this key step of coupling in literature three different synthetic strategies are known which foresee an exclusively enzymatic approach, exclusively chemical or chemo-enzymatic.
As for the enzymatic pathways families of sialyltransferases and transialidases (enzymes that add the sialic acid to oligosaccharides in a strictly specific way) were used. Examples of this route of synthesis are reported in A. T. Beyer et al Adv. Enzymol., 1981, 52, 23-175, in J. Weinstein et al. J. Biol. Chem., 1982, 257, 13845-13853.
However several are the limitations in the use of these enzymes:    1) the limited availability of these enzymes    2) the need to synthesize the donor of activated substrate CMP-NeuAc, or PNP-NeuAc    3) the strict specificity of the sialyltransferases which reduces the flexibility in use for the synthesis of natural sialosides (Ichigawa Y. et al. Analytical Biochem 1992, 202, 215-238, S. Sabesan et al. J. Am. Chem. Soc., 1986, 108, 2068-2080, O. Hindsgaul et al J. Biol. Chem., 1991, 266, 17858-17862, H. J. Gross et al. Eur. J. Biochem. 1988, 177, 583-589).
As for the chemo-enzymatic pathways these include chemical synthesis of the acceptor and then enzymatic sialylation as in S. Sabesan et al., J. Am. Chem. Soc., 1986, 108, 2068-2080.
Focusing exclusively on chemical ways it is emphasized that the formation of the glycosidic bond with sialic acid is a reaction rather difficult because it is hampered by the fact that the donor is electronically and sterically hindered by the geminal carboxyl group. Moreover, the lack of functional group on C-3 rules out its anchimeric assistance for controlling stereochemistry and leads to the formation of by-products through the reaction of elimination; finally the formation of the binding with α configuration is thermodynamically disadvantaged in relation to anomeric effect. In an attempt to remedy these defects in the state of the art various strategies have been developed for the preparation of suitably activated sialic donors and acceptor with adequately protected hydroxyl groups that are reacted through various glycosylation methods.
Regarding the sialic donor it is to be emphasized that the complicated molecular architecture imparts a substantial degree of difficulty in its synthesis, protection and activation. The multifunctional nature (3 secondary hydroxyl groups) as well as the tertiary anomeric center, complicate the work of synthetic chemist. Currently to the state of the art is well known that the sialic donor can be activated as 2-xanthate (A. Marra et al., Carbohydr. Res., 1989, 187, 35), as 2-aryl sulphone (Y. Du et al, Carbohydr. Res., 1998, 308, 161), as 2-phosphite (R R Schmidt et al., Tetrahedron Lett., 1992, 33, 6123 or C H Wong et al., J. Am. Chem. Soc., 1992, 114, 8748) or as 2-halo derivative. Among all these groups the halogen derivatives are preferable as the phosphite and the thio derivatives require toxic reagents for their synthesis and not easy to handle in the industry. Among the halogens the chloro derivative is preferred as it is stable and easy to synthesize, in fact the bromo derivative is unstable and tends easily to eliminate and lead to anomeric mixtures during glycosilation reactions. The fluoro derivative requires a more elaborate synthesis than the chloro derivative and tends to form β glycosidic bonds. The chloro derivative would be the easiest donor to make and use.
Regarding to the structure of the donor, other synthetic routes, still more complex than previous ones, require the inclusion on the C-3 of sialic acid of a functional group that gives anchimeric assistance in the glycosilation reaction to prevent competitive elimination in 2,3 position. For this reason groups such as phenylthio or phenylselenium have been introduced (Y. Ito et al., Tetrahedron Lett., 1988, 29, 3987 or in L. O. Kononov et al., Tetrahedron Lett., 1997, 38, 1599). These pathways thus require multiple steps to obtain the active donor for the glycosylation reaction generally starting from 2,3-dehydro NeuAc with yields that vary according to the obtained specificity and the ease of purification of the intermediates.
For this reason for the synthesis of sialyloligosaccharides of formula (I) and in particular the 6′SL it would be preferable to employ efficiently a simple sialylderivative as the 2-chloro-donor of formula (II)
where P is a suitable protecting group, R1 is alkyl group and X is chlorine; (obtainable by methods reported in R. Kuhn et al., Chem. Ber., 1966, 99, 611, A. Marra et al., Carb. Res., 1989, 190, 317-322 and N F Byramova et al., Carb. Res, 1992, 237, 161-175) because it is also easily to synthesize on an industrial scale without the use of very toxic reactives, and it leads to the specific formation of α bonds in the glycosilation reactions. The use of this sialic donor, however, decreased significantly after its first applications and the state of the art addresses towards much more complex donors.
As for the acceptor activation for the synthesis of 6′SL, in the literature there are acceptors substituted with ether protective groups, for example benzyl groups whose removal, requiring an hydrogenation, is not easilymanageable (G. Pazynina et al. Tetrahedron Lett, 2002, 43, 8011-8013) and therefore difficult for industrial application.
α-glycosides of sialic acid were prepared by the Koenigs-Knorr reaction involving the use of Ag (I) as a promoter [Koenigs, W., Knorr, E. Chem. Ber., 1901, 34, 957] or by using the Helferich modification that uses Hg (II) as promoter [Helferich, B.; Zirner, J. Chem. Ber., 1962, 95, 2604]. The substrates chosen in these reactions are β-glycosyl-halides. Many variations of these classical methods are known which were designed to improve employment opportunities and yields. The main differences between these variations are related to the choice of counter anion of the metallic promoter. The most commonly used promoters are AgOTf, Ag2CO3, HgX2 (X=halide), and Hg(CN)2. In general it is known that Ag(I) promoters are more active and stereoselective (Pazynina G. et al. Tetrahedron Lett, 2002, 43, 8011-8013) but these should be used in large quantities (6-7 eq in relation to the acceptor) thus increasing the cost of synthesis (including the disposal of waste production), while Hg(II) promoters provide higher yields (H. Paulsen et al. Angew. Chem. Int. Ed. Engl, 1982, 927-928) but can present difficulties in handling due to their toxicity.
It is therefore evident the need for a process for the synthesis of compounds of formula (I) which is simple and economical, applicable on industrial scale, and hence allowing to overcome the technical problems above mentioned related to processes known in the literature.